Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands

Abstract

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

Keywords: Culex cedecei; Everglades virus; evolution; phylogenetics; phylogeography.

Figure 1.

Map of southern Florida showing locations of EVEV samples from public lands. Circles indicate the current study, and diamonds indicate previous studies. Public lands include Florida Panther National Wildlife Refuge (FPNR), Fakahatchee Strand State Park Preserve (FAK), Big Cypress Preserve (BICY), and Everglades National Park (ENP). Arrows indicate approximate hurricane paths of Hurricane Andrew in 1992 and Hurricane Irma in 2017, both affecting South Florida.

Figure 2.

Spatiotemporal spread of EVEV in the Greater Everglades and position of unique mutations on E1. (A) MCC time-scaled phylogeny inferred by enforcing a strict clock and constant demographic prior in BEAST 1.10.4. Time of coalescent events (TMRCA) and 95 per cent HPD intervals (HPD) at major divergent event nodes are reported near nodes. The pie chart, at nodes for which the posterior probability was greater than 0.9, shows the ancestral state reconstruction probability for the origin. Branch lengths are scaled in time and colored according to location based on the legend in the figure. Mutations of relevance that number is the amino acid position of the protein where the mutation was mapped in the tree. (B) Mutations of interest are shown by green spheres on the E1 protein using the structural data available for the Capsid, E1, and E2 structure of the VEEV TC-83 strain (PDB ID: 3J0C). The black sphere indicated Position 226, site of the A226V mutation in E1 that allowed CHIKV to expand the host range.